At present, age-related macular degeneration (AMD) is one of the major causative diseases of legal blindness in advanced countries, and mainly seen in elderly citizens over 50 years of age. Age-related macular degeneration is caused by age-related changes in the macula, and largely divided into an exudative form and an atrophic form. Exudative age-related macular degeneration is a disease associated with the development of neovascular vessels from the choroid, in the macula of elderly citizens, then bleeding and exudative lesion under the retinal pigment epithelium or retina, and finally, formation of a scar tissue. Atrophic age-related macular degeneration is a disease accompanied by atrophy of the macular area and accumulation of drusen. A precursor lesion leading to exudative and atrophic age-related macular degeneration is sometimes referred to particularly as early age-related macular degeneration, and this lesion is also considered to be one pathology of age-related macular degeneration.
For the treatment of age-related macular degeneration when it is a mild exudative form, a surgical therapy such as photodynamic therapy, laser photocoagulation, stripping of neovascular vessel and the like, or a treatment method aiming at regression and removal of neovascular vessel by a drug therapy such as administration of an inhibitor of the vascular endothelial growth factor (VEGF) involved in the angiogenesis and the like can be selected. However, the aforementioned means cannot provide therapeutic effectiveness for the exudative or atrophic form that has progressed to the advanced atrophy or lack of retinal pigment epithelium (RPE). In such case, an effective treatment method is transplantation of retinal pigment epithelial cells or retinal pigment epithelium to the deficient site under the retina.
Various treatment methods by the transplantation of retinal pigment epithelial cells have been tried. It has been reported that transplantation of retinal pigment epithelial cells obtained from human fetus in the form of a cell suspension shows poor engraftment of the transplanted cells. In cases where a retinal pigment epithelial cell sheet also containing Bruch's membrane and choroid detached like a sheet from a cadaveric eye was transplanted, post-operative rejection occurred and a sufficient treatment effect was not obtained (non-patent document 1). In addition, the method utilizing a cadaveric eye has been pointed out to include an ethical problem. On the other hand, a case of improved eyesight of a patient with age-related macular degeneration has been reported, wherein used was a method including collecting the patient's own normal retinal pigment epithelial cells and choroid and transplanting them to the deficient site under the macula. However, this method places an extremely high burden on patients and an extremely high risk of surgery. While an attempt has been made to utilize the patient's own iris pigment epithelial cells collected from the patient with age-related macular degeneration for the transplantation instead of retinal pigment epithelial cells, the upper limit of the final eyesight is low and a sufficient effect has not been obtained, and moreover, the burden and risk of utilizing the patient's own cell are high (non-patent document 2). Thus, conventional treatment methods using cells collected from a cadaveric eye or the patient's own cells are not practical in terms of ethics, safety, effect and the like, and a retinal pigment epithelial cell truly usable for a transplantation treatment has been desired.
As one of the production methods of a retinal pigment epithelial cell sheet, a method including coating a cell culture substrate with an extracellular matrix and the like, and culturing retinal pigment epithelial cells thereon to form a sheet can be mentioned. For example, a cell sheet can be formed by culturing retinal pigment epithelial cells on a fibronectin-coated material, but formation of a basement membrane is not reported, and a method of taking out a sheet from a container in which the sheet was formed is not described at all (non-patent document 3). In consideration of such problems, a method of forming, in advance, a retinal pigment epithelial cell sheet by using an artificial membrane in place of a basement membrane has been developed by plural laboratories. However, an artificial membrane is feared to be a disorder for engraftment in transplantation and functional maintenance of the body. While an artificial membrane with a less biological response has also been developed, it is not suitable for transplantation since it problematically induces inflammation and rejection associated therewith, which are caused by the differences from the basement membrane produced by the cell itself in the composition, properties, rigidity and the like. Conventionally, moreover, a method of culturing a retinal pigment epithelial cell sheet having polarity has been known. However, it is only for experimental model use wherein retinal pigment epithelial cells and immortalized line derived from a living organism are/is made into a sheet in a container and directly utilized, and it has not yet enabled actual recovery of a retinal pigment epithelial cell sheet provided with a basement membrane as in living organisms from a container (non-patent document 4). There is a method of obtaining a retinal pigment epithelial cell sheet by exfoliating retinal pigment epithelial cells directly cultured on a culture dish, without using an extracellular matrix and the like. Such method of detaching cells by exfoliation cannot stabilize the shape and size of the sheet, basement membrane and the like, causes much damage on the retinal pigment epithelial cells by exfoliation, and causes, in most cases, disintegration of the shape of the sheet and scattered cells due to the exfoliation, thus failing to provide a cell sheet usable for transplantation and the like (non-patent document 5). In addition, while coating a cell culture substrate with collagen and culturing cells on the collagen has been reported, human retinal pigment epithelial cells are not used. Moreover, to increase the gel strength, a crosslinking agent needs to be added separately, which makes the method far from convenient and rapid (patent document 1). As the situation now stands, production of a retinal pigment epithelial cell sheet, which is therapeutically effective for retinal diseases such as age-related macular degeneration and the like, and convenient and stable to prepare, has still been a problem.